The fibrotic scar that results after a myocardial infarction (MI) is stiff extracellular matrix (ECM), owing to the enhanced secretion of collagen as the tissue thins and undergoes necrosis. Pervious methods to improve myocardial function post-MI, e.g. cardiac patches and cell injections, do not sufficiently mimic the intrinsic properties of the matrix, such as stiffness (denoted E). Moreover, they often employ adult stem cells which have not been shown to have significant remodeling capacity and instead are more responsive to aberrant matrix conditions; thus cells have been observed to improperly differentiate into osteoblast-like cells or to fail to differentiate altogether in infarcted myocardium that is 3-fold too stiff, i.e. EInfarct >> ECARDIO. We have recently developed a dynamic, thiol-modified hyaluronic acid (HA-S)-based hydrogel that displays developmentally appropriate stiffness over time via time-dependent crosslinking. We have also shown that this can improve cardiac progenitor differentiation in mature cardiomyocytes by nearly an order of magnitude over soft matrix that does not remodel. In this proposal, we will first extend our findings to embryonic stem cells (ESCs), which should be even more effective at matrix remodeling than previous stem cell types. Expression of cardiac-specific genes in 2D and 3D HA-S hydrogels will first be monitored in ESCs to determine if HA-S can induce cardiomyogenesis relative to cardiac progenitor cells and age-matched control animals, and if not, at least ensure that it enhances differentiation over HA-S hydrogels that have had their time-dependent crosslinking removed by treatment with iodacetamide. Matrix secretion, assembly, and remodeling (via degradation by hyaluronidase) will also be measured and compared to cardiac progenitor cells to ensure that ESCs can indeed remodel matrix effectively and that cells can migrate sufficiently in the material. Cells and HA-S hydrogels will subsequently be used in a subcutaneous rat model to ensure biocompatibility and monitor hydrogel properties in vivo, e.g. time-dependent stiffening. Finally in a rat model of MI, ESCs and/or cardiac progenitor will be used in conjunction with the HA-S to determine to what degree our HA-S hydrogel can improve myocardial function post-MI versus convention treatments, e.g. cell injection.